Over the years, it has been possible to characterize the composition of individual synthetic polymers of interest. This characterization usually has involved measuring the degree of polymerization, for example, measuring the number of particular primary units (building blocks), within a polymer of interest. This type of characterization may be adequate when the polymer is a synthetic polymer, for example, polyethylene, polypropylene, or the like, which exists as a mixture of individual polymer components made up of the same repeating unit (monomer), but having different degrees of polymerization. Because molecular weight reflects differences in the degree of polymerization, molecular weight alone may be sufficient to characterize polymeric mixtures made up of the same repeating units.
However, the characterization of complex polymeric mixtures, for example, polymeric mixtures in which each polymer may be made up of different building blocks, has proven to be far more difficult. Such mixtures occur in nature and can include, for example, mixtures of biopolymers in a sample of interest. For example, many therapeutically effective proteins are glycosylated with a diverse group of carbohydrates. Accordingly, these glycosylated proteins, also known as glycoproteins, exist as complex mixtures of proteins having different glycosylation patterns. As a result, molecular weight distribution alone usually cannot accurately describe batch-to-batch variations in different glycoprotein preparations or confirm that one glycoprotein preparation is the bio-equivalent of another glycoprotein preparation.
Sequencing methods have been developed for characterizing proteins (see, for example, “Biochemistry,” Third Edition (1988), by Stryer, published by Freeman & Co., NY), nucleic acids (see, for example, Stryer (1988) supra), and polysaccharides (see, for example, U.S. Pat. No. 6,597,996 and U.S. Patent Application Publication No. US2003/0096281). However, these methods alone typically are insufficient to fully characterize each of the individual biopolymer species that are present in complex biopolymer mixtures. For example, the characterization of each of the polysaccharides in a complex mixture may require the isolation of each polysaccharide species present in the mixture prior to its sequencing using the methods described, for example, in U.S. Pat. No. 6,597,996. For many mixtures, species isolation can be impractical or even impossible. Even when the individual species present in a biological mixture can be physically isolated and characterized, the resulting characterization often does not provide insight into the active species within the mixture or the biological activity of the mixture.
Accordingly, the currently available methods for characterizing polymers are usually inadequate for characterizing complex biological mixtures. The need for new methods for characterizing complex biological mixtures is particularly evident in the pharmaceutical and biotechnology industries. For example, there are a variety of biologics—for example, glycoproteins such as interferon, erythropoietin, and the like; polysaccharides such as chondroitin sulfate, hyaluronan, heparin, and the like; and synthetic peptides such as copolymer 1, and the like—that have been approved by the U.S. Food and Drug Administration for use in humans. However, a complete characterization of each of the polymers within the biologic may be helpful so as to minimize batch-to-batch variations between different preparations of the biologic or to produce a bio-equivalent preparation of a biologic already approved for use in humans.
Accordingly, there is an ongoing need for methods capable of characterizing the composition of complex biological mixtures.